(A) EMSA study. Both C/EBPɛ and C/EBP bound to the C/EBP site of the promoter of the G-CSF receptor. A ³²P γ-ATP–labeled, double-stranded oligonucleotide containing the C/EBP site of the promoter of the G-CSF receptor was added to lysate from cos-1 cells expressing either C/EBPɛ or C/EBP. Binding was competed by 20-fold excess of cold-self. Retarded bands were supershifted by specific antisera. (B) EMSA study with ³²P γ-ATP–labeled C/EBP consensus site oligonucleotide derived from neutrophil elastase promoter. Oligonucleotides were retarded by C/EBP protein complex present in nuclear extracts of the Kcl22 cell line in characteristic location for C/EBPɛ migration. Antibodies directed against C/EBPɛ (lane 7) and CRP1 (rat C/EBPɛ) (lane 8) either supershifted or blocked binding of the complex, respectively. Antibodies to C/EBP, C/EBPβ, and C/EBPδ did not supershift or block formation of the complex (lanes 9 through 11), indicating that C/EBPɛ is the predominant C/EBP protein binding to neutrophil elastase C/EBP consensus site in Kcl22 nuclear extracts.